BACKGROUND. RNAi is a powerful genetic tool for dissecting functional vulnerabilities in cancer cells. Currently, RNAi analysis has mostly been confined to single-gene phenotype studies. In human cells, the presence of functionally redundant gene paralogs and the complex interaction among Ras effector pathways requires a new approach to understand the function of signaling nodes from a systems biology perspective. PURPOSE. In this project we aim to accomplish the following goals: 1) constructing a high quality, knockdown validated siRNA library that target Ras effector genes; 2) using this siRNA library to analyze a panel of KRAS mutant and WT human cancer cell lines to identify common onco-effector dependencies in KRAS mutant cell lines; and 3) dissecting the mechanisms of onco-effect dependency in KRAS mutant cells using molecular and pharmacological tools. SIGNIFICANT MATERIALS AND METHODS. Knockdown validated siRNA library targeting Ras pathway genes. ACCOMPLISHMENT. We have identified and validated a set of siRNAs against Ras pathway genes. We demonstrated that these siRNAs can be used in a combinatorial setting to knockdown multiple genes. We have systematically evaluated siRNA combinations targeting single gene node and gene node pairs among major Ras effector pathways. We have identified RAF kinases as key Ras onco-effectors and the autophagy pathway as a critical co-dependency non-oncogene addiction pathway in KRAS mutant cells. We demonstrated that BRAF, CRAF and ATG7 could potentially be a new target combination for the treatment of KRAS mutant tumors. We have published two papers from this project.